Objective To investigate the physicochemical properties, osteogenic properties, and osteogenic ability in rabbit model of femoral condylar defect of acellular dermal matrix (ADM)/dicalcium phosphate (DCP) composite scaffold. Methods ADM/DCP composite scaffolds were prepared by microfibril technique, and the acellular effect of ADM/DCP composite scaffolds was detected by DNA residue, fat content, and α-1, 3-galactosyle (α-Gal) epitopes; the microstructure of scaffolds was characterized by field emission scanning electron microscopy and mercury porosimetry; X-ray diffraction was used to analyze the change of crystal form of scaffold; the solubility of scaffolds was used to detect the pH value and calcium ion content of the solution; the mineralization experiment in vitro was used to observe the surface mineralization. Twelve healthy male New Zealand white rabbits were selected to prepare the femoral condylar defect models, and the left and right defects were implanted with ADM/DCP composite scaffold (experimental group) and skeletal gold? artificial bone repair material (control group), respectively. Gross observation was performed at 6 and 12 weeks after operation; Micro-CT was used to detect and quantitatively analyze the related indicators [bone volume (BV), bone volume/tissue volume (BV/TV), bone surface/bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), bone mineral density (BMD)], and HE staining and Masson staining were performed to observe the repair of bone defects and the maturation of bone matrix. Results Gross observation showed that the ADM/DCP composite scaffold was a white spongy solid. Compared with ADM, ADM/DCP composite scaffolds showed a significant decrease in DNA residue, fat content, and α-Gal antigen content (P<0.05). Field emission scanning electron microscopy showed that the ADM/DCP composite scaffold had a porous structure, and DCP particles were attached to the porcine dermal fibers. The porosity of the ADM/DCP composite scaffold was 76.32%±1.63% measured by mercury porosimetry. X-ray diffraction analysis showed that the crystalline phase of DCP in the ADM/DCP composite scaffolds remained intact. Mineralization results in vitro showed that the hydroxyapatite layer of ADM/DCP composite scaffolds was basically mature. The repair experiment of rabbit femoral condyle defect showed that the incision healed completely after operation without callus or osteophyte. Micro-CT showed that bone healing was complete and a large amount of new bone tissue was generated in the defect site of the two groups, and there was no difference in density between the defect site and the surrounding bone tissue, and the osteogenic properties of the two groups were equivalent. There was no significant difference in BV, BV/TV, BS/BV, Tb.Th, Tb.N, and BMD between the two groups (P>0.05), except that the Tb.Sp in the experimental group was significantly higher than that in the control group (P<0.05). At 6 and 12 weeks after operation, HE staining and Masson staining showed that the new bone and autogenous bone fused well in both groups, and the bone tissue tended to be mature. Conclusion The ADM/DCP composite scaffold has good biocompatibility and osteogenic ability similar to the artificial bone material in repairing rabbit femoral condylar defects. It is a new scaffold material with potential in the field of bone repair.
ObjectiveTo explore the feasibility of chitosan/allogeneic bone powder composite porous scaffold as scaffold material of bone tissue engineering in repairing bone defect.
MethodsThe composite porous scaffolds were prepared with chitosan and decalcified allogeneic bone powder at a ratio of 1∶5 by vacuum freeze-drying technique. Chitosan scaffold served as control. Ethanol alternative method was used to measure its porosity, and scanning electron microscopy (SEM) to measure pore size. The hole of 3.5 mm in diameter was made on the bilateral femoral condyles of 40 adult Sprague Dawley rats. The composite porous scaffolds and chitosan scaffolds were implanted into the hole of the left femoral condyle (experimental group) and the hole of the right femoral condyle (control group), respectively. At 2, 4, 8, and 12 weeks after implantation, the tissues were harvested for gross observation, histological observation, and immunohistochemical staining.
ResultsThe composite porous scaffold prepared by vacuum freeze-drying technique had yellowish color, and brittle and easily broken texture; pore size was mostly 200-300μm; and the porosity was 76.8%±1.1%, showing no significant difference when compared with the porosity of pure chitosan scaffold (78.4%±1.4%) (t=-2.10, P=0.09). The gross observation and histological observation showed that the defect area was filled with new bone with time, and new bone of the experimental group was significantly more than that of the control group. At 4, 8, and 12 weeks after implantation, the bone forming area of the experimental group was significantly larger than that of the control group (P < 0.05). The immunohistochemical staining results showed that osteoprotegerin (OPG) positive expression was found in the experimental group at different time points, and the positive expression level was significantly higher than that in the control group (P < 0.05).
ConclusionChitosan/allogeneic bone powder composite porous scaffold has suitable porosity and good osteogenic activity, so it is a good material for repairing bone defect, and its bone forming volume and bone formation rate are better than those of pure chitosan scaffold.
ObjectiveTo review the application of silk fibroin scaffold in bone tissue engineering.
MethodsThe related literature about the application of silk fibroin scaffold in bone tissue engineering was reviewed, analyzed, and summarized.
ResultsSilk fibroin can be manufactured into many types, such as hydrogel, film, nano-fiber, and three-dimensional scaffold, which have superior biocompatibility, slow biodegradability, nontoxic degradation products, and excellent mechanical strength. Meanwhile these silk fibroin biomaterials can be chemically modified and can be used to carry stem cells, growth factors, and compound inorganic matter.
ConclusionSilk fibroin scaffolds can be widely used in bone tissue engineering. But it still needs further study to prepare the scaffold in accordance with the requirement of tissue engineering.
ObjectiveTo investigate the influences of lactic acid (LA), the final degradation product of polylactic acid (PLA) on the prol iferation and osteoblastic phenotype of osteoblast-l ike cells so as to provide theoretical basis for bone tissue engineering.
MethodsRos17/2.8 osteoblast-l ike cells were harvested and divided into 3 groups. In groups A and B, the cells were cultured with the medium containing 4, 8, 16, 22, and 27 mmol/L L-LA and D, L-LA, respectively. In group C, the cells were cultured with normal medium (pH7.4). The cell prol iferation was determined with MTT method after 1, 3, and 5 days. The relative growth ratio (RGR) was calculated, and the cytotoxicity was evaluated according to national standard of China. In addition, the alkal ine phosphatase (ALP) activity of cells cultured with medium containing 4 mmol/L L-LA (group A), 4 mmol/ L D, L-LA (group B), and normal medium (group C) after 1 and 5 days were detected with ALP kits, and the relative ALP ratio (RAR) was calculated; after 21 days, the calcium nodules were tested with von Kossa staining method, and were quantitatively analyzed.
ResultsWhen LA concentration was 4 mmol/L, the mean RGR of both groups A and B were all above 80%, and the cytotoxic grades were grade 0 or 1, which meant non-cytotoxicity. When LA concentration was 8 mmol/L and 16 mmol/ L, groups A and B showed cytotoxicity after 5 days and 3 days, respectively. When LA concentration was above 22 mmol/L, cell prol iferations of groups A and B were inhibited evidently after 1-day culture. At each LA concentration, RGR of group A was significantly higher than that of group B at the same culture time (P<0.05) except those at 4 mmol/L after 1-day and 3-day culture. After 1 day, the RAR of group A was significantly higher than that of group B on 1 day (144.1%±3.2% vs. 115.2%±9.8%, P<0.05) and on 5 days (129.6%±9.8% vs. 78.2%±6.9%, P<0.05). The results of von Kossa staining showed that the black gobbets in group A were obviously more than those of groups B and C. The staining area of group A (91.2%±8.2%) was significantly higher than that of groups B (50.3%±7.9%) and C (54.2%±8.6%) (P<0.05).
ConclusionThe concentration and composition of LA have significant effects on the cell proliferation and osteoblastic phenotype of osteoblast-l ike cells.
Objective To find a new culture system to induce proliferation and osteodifferentiation of marrow stromal cells (MSCs) in vitro for bone tissueeng ineering． Methods There were four groups in this experiment to study effects of Passage 3 osteoblasts derived from the rat cranium and the osteogenic inductor (1 nmol/L dexamethasone，10 mmol/L beta-glycero-phosphate，50 μg/ml retin oic acid) on growth of MSCs isolated from the rat femur and the tibia． MSCs were cultured in the DMEM medium (the c ontrol group) and in the osteoinductive culture medium (the inductor group)；fur thermore, MSCs were co-cultured with the osteoblasts in the DMEM medium (the osteoblast group) and in the osteoinductive culture medium (the combined treatment group)．The cells in the four groups were counted every 2 days for 8 days and alkaline phosphatase (ALP) activity of MSCs at 10 days of cultivation was measured．The MRNA expression of osteocalcin (OC) of MSCs at 2 weeks was assayed with the reverse transcript polymase chain reaction (RT-PCR)． Results There were more cells in the osteoblast group than in the control group(31.73±3.31×104 V S. 24.33±3.04×104, Plt;0.05), but there were fewer cells in the inductor gro up(16.23±2.44×104, Plt;0.05). There was no significant difference in th e cell number between the combined treatment group (21.54±2.29×104) and th e control group（Pgt;0.05）．The ALP activity was higher in the combined trea tment group （2.01±0.56 U）than in the control group (1.27±0.43 U), in the inductor group（1.27±0.43 U）, and in the osteoblast group （0.77±0.19 U）.The osteocalcin mRNA was expressed in the three treat ment groups but was not expressed in the control group. The significantly higher leve l of the osteocalcin mRNA was expressed in the inductor group（0.783±0.094）and in the combined treatment group（0.814±0.071）than in the osteoblast group（0.302±0.026） (Plt;0.05). Conclusion The combined use of t he osteoblast and the inductor can induce marrow stromal cells. Their combined u se does not affect the normal proliferation but can obviously promote the osteodifferentiation of marrow stromal cells. This combined use can become a new culture system of the seed cells for bone tissue engineering．
Objective To investigate the ectopic bone formation of the chitosan/phosphonic chitosan sponge combined with human umbil ical cord mesenchymal stem cells (hUCMSCs) in vitro. Methods Phosphorous groups were introduced in chitosan molecules to prepare the phosphonic chitosan; 2% chitosan and phosphonic chitosan solutions were mixed at a volume ratio of 1 ∶ 1 and freeze-dried to build the complex sponge, and then was put in the simulated body fluid for biomimetic mineral ization in situ. The hUCMSCs were isolated by enzyme digestion method from human umbil ical cord and were cultured. The chitosan/phosphonic chitosan sponge was cultured with hUCMSCs at passage 3, and the cell-scaffoldcomposite was cultured in osteogenic medium. The growth and adhesion of the cells on the scaffolds were observed by l ight microscope and scanning electron microscope (SEM) at 1 and 2 weeks after culturing, respectively. The cell prol iferation was detected by MTT assay at 1, 2, 3, 4, 5, and 6 days, respectively. Bilateral back muscles defects were created on 40 New Zealand rabbits (3-4 months old, weighing 2.1-3.2 kg, male or female), which were divided into groups A, B, and C. In group A, cellscaffold composites were implanted into 40 right defects; in group B, the complex sponge was implanted into 20 left defects; and in group C, none was implanted into other 20 left defects. The gross and histological observations were made at 4 weeks postoperatively. Results The analysis results of phosphonic chitosan showed that the phosphorylation occurred mainly in the hydroxyl, and the proton type and chemical shifts intensity were conform to its chemical structure. The SEM results showed that the pores of the chitosan/phosphonic chitosan sponge were homogeneous, and the wall of the pore was thinner; the coating of calcium and phosphorus could be observed on the surface of the pore wall after mineral ized with crystal particles; the cells grew well on the surface of the chitosan/phosphonic chitosan sponge. The MTT assay showed that the chitosan/phosphonic chitosan sponge could not inhibit the prol iferation of hUCMSCs. The gross observation showed that the size and shape of the cell-scaffold composite remained intact and texture was toughened in group A, the size of the complex sponge gradually reducedin group B, and the muscle defects wound healed with a l ittle scar tissue in group C. The histological observation showed that part of the scaffold was absorbed and new blood vessels and new bone trabeculae formed in group A, the circular cavity and residual chitosan scaffolds were observed in group B, and the wound almost healed with a small amount of lymphocytes in group C. Conclusion The chitosan/phosphonic chitosan sponge has good biocompatibil ity, the tissue engineered bone by combining the hUCMSCs with chitosan/phosphonic chitosan sponge has the potential of the ectopic bone formation in rabbit.
Objective To review the research progress on bone repair biomaterials with the function of recruiting endogenous mesenchymal stem cells (MSCs). Methods An extensive review of the relevant literature on bone repair biomaterials, particularly those designed to recruit endogenous MSCs, was conducted, encompassing both domestic and international studies from recent years. The construction methods and optimization strategies for these biomaterials were summarized. Additionally, future research directions and focal points concerning this material were proposed. Results With the advancement of tissue engineering technology, bone repair biomaterials have increasingly emerged as an ideal solution for addressing bone defects. MSCs serve as the most critical “seed cells” in bone tissue engineering. Historically, both MSCs and their derived exosomes have been utilized in bone repair biomaterials; however, challenges such as limited sources of MSCs and exosomes, low survival rates, and various other issues have persisted. To address these challenges, researchers are combining growth factors, bioactive peptides, specific aptamers, and other substances with biomaterials to develop constructs that facilitate stem cell recruitment. By optimizing mechanical properties, promoting vascular regeneration, and regulating the microenvironment, it is possible to create effective bone repair biomaterials that enhance stem cell recruitment. Conclusion In comparison to cytokines, phages, and metal ions, bioactive peptides and aptamers obtained through screening exhibit more specific and targeted recruitment functions. Future development directions for bone repair biomaterials will involve the modification of peptides and aptamers with targeted recruitment capabilities in biological materials, as well as the optimization of the mechanical properties of these materials to enhance vascular regeneration and adjust the microenvironment.
ObjectiveTo review the progress of cell sheet technology (CST) and its application in bone tissue engineering. MethodsThe literature concerning CST and its application was extensively reviewed and analyzed. ResultsCST using temperature-responsive culture dishes is applied to avoid the shortcomings of traditional tissue engineering. All cultured cells are harvested as intact sheets along with their deposited extracellular matrix. Avoiding the use of proteolytic enzymes, cell sheet composed of the cells and extracellular matrix derived from the cells, and remained the relative protein and biological activity factors. Consequently, cell sheet can provide a suitable microenvironment for the bone regeneration in vivo. With CST, cell sheet engineering is allowed for tissue regeneration by the creation of three-dimensional structures via the layering of individual cell sheets, be created by wrapping scaffold with cell sheets, or be created by folding the cell sheets, showing great potential in tissue engineered bone. ConclusionConstructing tissue engineered bone using CST and traditional method of bone tissue engineering will promote the development of the bone tissue engineering.
ObjectiveTo study the preparation and cytocompatibility of bone tissue engineering scaffolds by combining low temperature three dimensional (3D) printing and vacuum freeze-drying techniques.
MethodsCollagen (COL)and silk fibroin (SF) were manufactured from fresh bovine tendon and silkworm silk. SolidWorks2014 was adopted to design bone tissue engineering scaffold models with the size of 9 mm×9 mm×3 mm and pore diameter of 500μm. According to the behavior of composite materials that low temperature 3D printing equipment required, COL, SF, and nano-hydroxyapatite (nHA)at a ratio of 9:3:2 and low temperature 3D printing in combination with vacuum freeze-drying techniques were accepted to build COL/SF/nHA composite scaffolds. Gross observation and scanning electron microscope (SEM) were applied to observe the morphology and surface structures of composite scaffolds. Meanwhile, compression displacement, compression stress, and elasticity modulus were measured by mechanics machine to analyze mechanical properties of composite scaffolds. The growth and proliferation of MC3T3-E1 cells were evaluated using SEM, inverted microscope, and MTT assay after cultured for 1, 7, 14, and 21 days on the composite scaffolds. The RT-PCR and Western blot techniques were adopted to detect the gene and protein expressions of COL I, alkaline phosphatase (ALP), and osteocalcin (OCN) in MC3T3-E1 cells after 21 days.
ResultsCOL/SF/nHA composite scaffolds were successfully prepared by low temperature 3D printing technology and vacuum freeze-drying techniques; the SEM results showed that the bionic bone scaffolds were 3D polyporous structures with macropores and micropores. The mechanical performance showed that the elasticity modulus was (344.783 07±40.728 55) kPa; compression displacement was (0.958 41±0.000 84) mm; and compression stress was (0.062 15±0.007 15) MPa. The results of inverted microscope, SEM, and MTT method showed that a large number of cells adhered to the surface with full extension and good cells growth inside the macropores, which demonstrated a satisfactory proliferation rate of the MC3T3-E1 cells on the composite scaffolds. The RT-PCR and Western blot electrophoresis revealed gene expressions and protein synthesis of COL I, ALP, and OCN in MC3T3-E1 cells.
ConclusionLow temperature 3D printing in combination with vacuum freeze-drying techniques could realize multi-aperture coexisted bionic bone tissue engineering scaffolds and control the microstructures of composite scaffolds precisely that possess good cytocompatibility. It was expected to be a bone defect repair material, which lays a foundation for further research of bone defect.
Objective Rapid and effective vascularization of scaffolds used for bone tissue engineering is critical to bony repair. To study the cooperative and promotion effects of enhanced bioactive glass/collagen composite scaffold on vascularization for searching for a kind of el igible vascularized scaffold to repair bone defect. Methods The human umbil ical vein endothel ial cells (HUVECs) were collected from human umbil ical core, and identified through von Willebrandfactor (vWF) and CD34 immunofluorescence. The 1st passage of HUVECs were suspensed and seeded into the scaffold. The attachment and prol iferation of HUVECs on the scaffold were observed through scanning electron microscope (SEM). HUVECs were seeded on the scaffold as the experimental group, and on 96-well plate as the control group. The growth rate of HUVECs was detected through alarmarBlue at 1, 3, 5, 7, 9, and 11 days. Meanwhile, the mRNA expression levels of VEGF, fms-related tyrosine kinase 1 (Flt-1), and kinase insert domain receptor (Kdr) were detected through real-time fluorescence quantitative PCR. Twelve scaffolds were embedded subcutaneouly into 6 Sprague-Dawley rats. The enhanced scaffolds were used and the arteria and vein saphena bundle were embedded straightly through the central slot of scaffold in experimental group, and the common scaffolds were used in control group. Frozen section and HE staining of scaffolds were performed at 5 days and 10 days to observe the vascularization of embedded scaffold. Results HUVECs were identified through morphology, vWF and CD34 immunofluorescence. SEM results showed HUVECs could attach to the scaffold tightly and viably. HUVECs prol iferated actively on the scaffold in experimental group; the growth rate in experimental group was higher than that in control group at 3-11 days, showing significant differences within 5-11 days (P lt; 0.05). The real-time fluorescence quantitative PCR results showed thatthe mRNA expression levels of VEGF, Flt-1, and Kdr in experimental group were higher than those in control group at 3 days, showing significant differences (P lt; 0.05). Frozen section and HE staining of the scaffolds in experimental group showed that the embedded vessel bundle were still patency at 5 days and 10 days, that many new vessels were observed around the embedded vessel bundle and increased with time, host vessels infiltrated in the surrounding area of scaffold and fewer neo-vessels at the distant area. But there was only some fibrous tissue appeared in control group, and at 10 days, the common scaffold degradated, so few normal tissue appeared at the embedded area. Conclusion Enhanced bioactive glass/collagen composite scaffold can promote vascularization in vitro and in vivo, and may be used in bone tissue engineering.
